Human papillomaviruses (HPVs) are the most common sexually transmitted infection. These viruses infect and replicate in mucosal and cutaneous epithelium, inducing cell proliferation as part of their replicative life cycle. The high risk oncogenic HPVs cause 250,000 cases of cervical cancer worldwide, the second most common cancer in women. HPVs are small DNA viruses and must deliver their genomes to the host cell nucleus to initiate a successful infection. Like all other non-enveloped viruses, HPVs are faced with the task of transferring their genetic material (vDNA) across a limiting membrane, a critical event mediated by the minor capsid protein L2. Recent studies have implicated the N-terminal domain of L2 as a crucial region for this membrane penetration activity. N-terminal cleavage of L2 by the host cell protease furin and a conserved disulfide bond between Cys22 and Cys28 are essential for vDNA translocation and we recently identified and implicated an N-terminal transmembrane domain (TMD) in this process as well. Herein, we propose studies aimed at understanding L2-dependent vDNA translocation, specifically aimed at developing a structural and mechanistic comprehension of the roles of furin cleavage, the disulfide bond, and the TMD of L2, in addition to the involvement of interacting cellular factors. We will use a variety of structural, biochemical, and cell-based experimental approaches to gain knowledge about this poorly understood process. These proposed studies will further advance our understanding of processes critical to the infectious life cycle of the oncogenic HPVs and thus represent potential drug targets for the development of low cost anti-HPV prophylactics that could be applied on condoms or within lubricants to further decrease transmission of these potentially deadly viruses. Certain viruses and bacterial toxins are known to possess these membrane penetration capabilities but their reliance on specific host cellular proteins suggest it is possibe that endogenous cellular proteins could utilize similar pathways. Thus, these studies have potential to unveil novel pathways and transport mechanisms in cell biology. Lastly, knowledge of the specific mechanisms of vDNA translocation will also be important towards understanding if and how host cells sense infection by HPVs through activation of innate immune response pathways during viral invasion and membrane penetration. These fundamental processes may therefore contribute towards viral evasion of early innate immune responses and could influence the establishment of persistent infection--hallmarks of oncogenic HPVs and a feature that undoubtedly contributes to the oncogenic nature of these viruses.